Anesthesiologists routinely select from a host of chemically diverse compounds to render patients unconscious and insentient, allowing painless performance of surgical procedures. Remarkably, however, the molecular and neuronal mechanisms by which most anesthetic drugs mediate their clinically important actions still remain uncertain. The development of new mouse genetic models in which candidate anesthetic targets can be disabled, either globally or in specific cell types, is beginning to reveal relative contributions of different molecular targets and cell groups to particular anesthetic actions. In this application, we employ such models to examine contributions of two subthreshold anesthetic-sensitive ion channels - HCN hyperpolarization- activated cation channels and TASK background potassium channels - in mediating clinically important anesthetic actions. The hypothesis guiding Specific Aim 1, which builds on recently published results from our laboratory, is that selective inhibition of dendritic HCN1 channels in cortical pyramidal neurons contributes to anesthetic-induced hypnosis. Proposed experiments focus primarily on HCN1-mediated actions of ketamine, a dissociative anesthetic for which a robust decrease in hypnotic sensitivity was obtained in conventional HCN1 knockout mice. Our aims are to: examine the role of pyramidal neurons in hypnotic anesthetic actions by using a forebrain-selective HCN1 knockout model; characterize effects of other dissociative anesthetics on HCN channels and anesthetic-induced hypnosis; identify molecular determinants for subunit-selective effects of ketamine on HCN1 channels; and develop/test a knock-in mouse model in which HCN1 channels are intact, but rendered insensitive to ketamine. The working model underlying Specific Aim 2, which derives from new preliminary data, is that inhibition of TASK channels by local anesthetics contributes to their deleterious effects. Our aims are to: evaluate pro-convulsive CNS effects of systemically administered local anesthetics in conventional TASK-1-/-:TASK-3-/- knockout mice; characterize relative sensitivity of TASK channels to different local anesthetics that vary in systemic toxicity; determine TASK channel contributions to local anesthetic action on excitatory thalamocortical circuit neurons; and examine specifically the role of those neurons in TASK channel-mediated CNS effects of local anesthetics by using cell- specific conditional TASK knockout mice. For both aims, we use a variety of approaches, including channel mutagenesis and patch clamp recordings from transfected cells; molecular and immunochemical validation of novel conditional mouse models; somatic and dendritic recordings from key neurons in wild type and mutant mice; and behavioral assessments of anesthetic sensitivity in those mice. The proposed studies provide a test of HCN and TASK channel subunit contributions to specific neuronal and behavioral actions of general and local anesthetics. Identification of these molecular and neural mechanisms may lead to discovery of safer, more effective anesthetic compounds, an important goal of anesthesia research. PUBLIC HEALTH RELEVANCE: General and local anesthetics remain among the most widely used and clinically useful drugs. Despite prevalent use and extensive clinical experience with these compounds, the molecular and neural mechanisms by which they mediate their desirable and untoward actions remain uncertain. The research undertaken in this proposal seeks to clarify those mechanisms, and thus to provide insights that may lead to development of more effective and safer anesthetic agents.